利用烟草表达人凝血IX因子(hFIX)的研究

Research on the Expression of Human Coagulation Factor IX in Tobacco

血友病B是凝血IX因子(hFIX)缺乏所导致的一种出血性疾病,通过输血和hFIX浓缩剂进行治疗疗效显著,但存在治疗费用高和安全隐患,因而获得安全、廉价的人凝血IX因子对血友病B治疗具有重要意义。植物系统表达外源蛋白在生产成本和安全性方面具有优势。为此,构建含人凝血IX因子基因(hFIX,2.8kb)植物双元表达载体p35s2300∷gus∷noster,用农杆菌介导法转化烟草“百日红”,通过PCR和Southernblot分析证实获得4株独立转基因植株,hFIX在转基因烟草基因组中的拷贝数为1～4个;RTPCR和ELISA检测结果表明,hFIX在转录和翻译水平已成功表达,hFIX在转基因烟草叶片中的表达量为2.5～8.8ng/g·FW,并具有免疫活性。为利用植物系统表达hFIX的后续研究作了必要准备,也为利用植物系统表达其他药用蛋白研究提供了一些理论和实验参考。

Hemophilia B is a bleeding coagulation protein factor IX, Currently treatment disorder caused by deficiency or abnormality in the plasma method of hemophilia B is based on protein replacement therapy by blood transfusion and intravenous infusion of condensate of coagulation protein factor IX with excellent therapy effect, which also has some problems including expensive costs, and high risks of virus infection by blood filtering. So how to obtain human factor IX more cheaply and safely is very important for the cure of hemophilia B. Plant expression system own advantage on cost and security, which showed good potential for production of human factor IX. Therefore plant binary expression vector p35S-2300 ：： hFIX（2.8kb） ：： noster was constructed and introduced into Agrobacterium tumefaciens strain EHA105 by freezing-melting method. The resulting vector was introduced into tobacco （Nicotiana tabacum L. cv. Bai Rihong ）plants via Agrobacterium tumefaciens-mediated method. Total 4 independent transgenic tobacco plants confirmed by PCR and Southern blot analysis were obtained. The hFIX gene was introduced into genome of transgenic tobacco plants, ranging from 1 to 4 copies. The expression of the introduced gene in transgenic plants was confirmed by RT-PCR and ELISA analysis at transcript and translation level, respectively. Content of hFIX ranged from 2.5 to 8.8ng/g· FW in fresh leaves of transgenic tobacco with immunocompetence. Good foundation for further expression of hFIX was providecl and also helpful for expression of other pharmaceutical proteins using plant expression system.